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PhosphoSens CDK4/CycD1 Protein Kinase Assays, Substrates & Recombinant Enzymes
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About CDK4/CycD1
Cyclin-dependent kinase 4 (CDK4) is a serine/threonine kinase belonging to the CDK family, which collectively governs cell cycle progression. CDK4 forms an obligate heterodimeric complex with its regulatory partner Cyclin D1 (encoded by CCND1), and this complex is the primary driver of G1-phase progression. Upon mitogenic stimulation, Cyclin D1 accumulates and binds CDK4, enabling phosphorylation of the retinoblastoma protein (RB1) at multiple sites, thereby releasing E2F transcription factors to activate S-phase gene programs. CDK4/CycD1 activity is antagonized by INK4 family inhibitors (p16/CDKN2A, p15, p18, p19) and is further regulated by CDK-activating kinase (CAK)-mediated T-loop phosphorylation. Dysregulation through CCND1 amplification, CDK4 amplification, or CDKN2A deletion is among the most frequent oncogenic events across cancers including breast, lung, melanoma, liposarcoma, and glioblastoma. The CDK4/RB axis represents a validated therapeutic target, with selective CDK4/6 inhibitors demonstrating profound clinical benefit, making robust biochemical assays essential for drug discovery and resistance mechanism studies.
Why PhosphoSens for CDK4/CycD1?
CDK4/CycD1 presents significant assay challenges: it requires co-expression and purification of the Cyclin D1 complex, exhibits relatively low intrinsic catalytic activity compared to other CDKs, and demands physiological ATP concentrations to accurately profile inhibitor selectivity against the CDK4/6 inhibitor class. Traditional endpoint methods such as ADP-Glo and HTRF obscure transient kinetic behavior, require low ATP concentrations that skew ATP-competitive inhibitor IC50 values, and cannot capture time-dependent inhibition relevant to covalent or slow-binding compounds. Radiometric assays add throughput and safety burdens. PhosphoSens continuous fluorescent kinase assays overcome these limitations by monitoring RB1-derived substrate phosphorylation in real time at physiological ATP, generating full progress curves that reveal enzyme activation lags, substrate depletion, and inhibitor mechanism of action, enabling accurate kinact/KI determination and reproducible SAR data for CDK4/6 inhibitor programs.
Key Pathways:
Cell Cycle G1/S Transition
RB/E2F Signaling
PI3K/AKT/mTOR Pathway
MAPK/ERK Mitogenic Signaling
Approved Inhibitors:
Palbociclib (Ibrance, HR+/HER2- breast cancer
Ribociclib (Kisqali, HR+/HER2- breast cancer
Abemaciclib (Verzenio, HR+/HER2- breast cancer and early-stage breast cancer)
Talk to a Scientist
Have questions about CDK4/CycD1 assay design, selectivity panels, or covalent inhibitor characterization?
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How pricing works:
Select a kit or substrate below, then request a quote. Bundle discounts apply automatically.
Select a kit or substrate below, then request a quote. Bundle discounts apply automatically.
Real-Time CDK4/CycD1 Activity Assays
Continuous, real-time fluorescent assays optimized for quantitative CDK4/CycD1 activity measurements, IC50 determination, and mechanistic studies.
Assay Format: Continuous kinetic fluorescence (real-time)
PhosphoSens-Kinetic assays directly quantify enzyme activity by continuously monitoring substrate phosphorylation or dephosphorylation in real time, generating a full progress curve in every well.
Learn more about PhosphoSens-Kinetic →Need pricing or availability? Select a kit or substrate to request a quote below.
Kits
PhosphoSens-Kinetic Kinase Activity Kit - AQT0258
Ready-to-use assay kits containing substrate and all essential reagents.
Kit Contents
- Optimized PhosphoSens® substrate (AQT0258)
- ATP, DTT, EGTA, Reaction Buffer & Dilution Buffer
Automatically save 10% when bundling 10ug recombinant enzyme with your 1,000 assay kit. View enzymes
Price: $425
Substrate
PhosphoSens Kinase Substrate AQT0258
Bulk PhosphoSens® substrate for assay development and high-throughput workflows.
Specifications
Automatically save 10% when bundling Reagent Packs with your substrate. View Reagent Packs
Price: $2795
Ready to request pricing for CDK4/CycD1?
Select a kit, substrate, or enzyme above. Our team will confirm pricing, availability, and any applicable bundle discounts.
Request a QuoteTRF CDK4/CycD1 Screening Assays
Robust endpoint TRF assays optimized for high-throughput screening and quantitative CDK4/CycD1 activity measurement in plate-based workflows.
Assay Format: Time-resolved fluorescence (TRF, endpoint)
PhosphoSens-Red assays use europium-based time-resolved fluorescence detection to enable robust, plate-based quantification of enzyme activity, making them well suited for high-throughput screening workflows.
Learn more about PhosphoSens-Red →Need pricing or availability? Select a kit or substrate to request a quote below.
Kits
PhosphoSens-Red Kinase Activity Kit - AQT0730
Ready-to-use TRF assay kits containing substrate and essential reagents.
Kit Contents
- Optimized PhosphoSens® substrate (AQT0730)
- ATP, DTT, EGTA, Reaction Buffer & Dilution Buffer
- Europium Detection Solution
Automatically save 10% when bundling 10ug recombinant enzyme with your 1,000 assay kit. View enzymes
Price: $455
Substrate
PhosphoSens Kinase Substrate AQT0730
Bulk substrate for PhosphoSens-Red TRF assays and high-throughput workflows.
Specifications
Automatically save 10% when bundling Reagent Packs with your substrate. View Reagent Packs
Price: $2795
Ready to request pricing for CDK4/CycD1?
Select a kit, substrate, or enzyme above. Our team will confirm pricing, availability, and any applicable bundle discounts.
Request a QuoteNo recombinant enzymes are currently available for CDK4/CycD1.
Frequently Asked Questions: CDK4/CycD1 Assays
Why is it important to run CDK4/CycD1 assays at physiological ATP concentrations?
CDK4/6 inhibitors such as palbociclib and abemaciclib are ATP-competitive, meaning their measured IC50 values are highly sensitive to ATP concentration via the Cheng-Prusoff relationship. Running assays at artificially low ATP skews potency data and poor predicts cellular activity. PhosphoSens enables real-time monitoring at physiological ATP (1-5 mM range), yielding IC50 and Ki values that translate more faithfully to cellular and in vivo settings.
Can PhosphoSens assays distinguish between CDK4 and CDK6 inhibitor selectivity?
Yes. Because PhosphoSens assays use kinase-specific peptide substrates derived from the RB1 phosphorylation site and generate continuous progress curves, subtle differences in inhibitor potency and kinetics between CDK4/CycD1 and CDK6/CycD3 can be resolved with high precision. Full progress curve analysis also reveals mechanistic differences, such as slow-on/slow-off binding kinetics, that endpoint assays would miss entirely.
How does the PhosphoSens assay handle the low activity of CDK4/CycD1 compared to other CDKs?
CDK4/CycD1 has intrinsically lower catalytic turnover than CDK2 or CDK1, which can make signal generation challenging in endpoint formats. PhosphoSens fluorescent substrates provide a continuous, sensitive readout that accumulates signal over the full reaction time course, amplifying detection sensitivity without requiring endpoint signal amplification steps or antibody-based detection, making it well-suited for low-activity kinase complexes.
Data & Resources
Explore data and documents to support your kinase and phosphatase experiments. Download sample data, protocols and other resources to see how our assays perform and to help you get started in your own lab. All validation data generated using PhosphoSens® assays under recommended conditions.
Sample Report: AQT0734 - EGFR Assay Validation Report
Each validation report provides experimental conditions and data showing:
- Enzyme titration and linearity
- Sensor peptide substrate titration and Km determination
- ATP titration and Km determination
- DMSO titration and assay tolerance
- Inhibitor titration and IC50 determination
Protocol
Inhibitor IC50 Protocol
See how the PhosphoSens-Kinetic Assay can be used to find the IC50 of a kinase inhibitor.
Download Protocol →Webinar
Webinar: Continuous Assay Validation
Discover how continuous assay formats power deep understanding of kinase function. See how PhosphoSens® assays guide inhibitor profiling, selectivity assessment, and mechanistic characterization.
Watch the Webinar →Service
KinSight™ CDK4/CycD1 Profiling
Need broader selectivity data? KinSight profiling runs your compounds across our full kinase panel under identical PhosphoSens conditions.
Learn about KinSight →Stay Informed
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